Project Summary. Enterotoxigenic E. coli (ETEC) is a food and waterborne pathogen that constitutes a bioterrorism and public health threat of global proportion for which no vaccine exists. We propose to develop an adhesin-toxoid chimera vaccine, exploiting the previous work of the partners on each of the two components. We have shown the feasibility of genetically combining genes to express a protein chimera consisting of the binding (B) subunit of cholera toxin and a fusion of CfaE, the minor adhesive subunit of colonization factor antigen I (CFA/I), to the CT A2 anchoring domain (where the adhesin replaces the toxic enzymatic domain of the CT A subunit). This chimera elicits strong humoral immune responses upon mucosal immunization of mice, and antibodies inhibit ETEC adherence in vitro. Our aims are to optimize the CfaE-CTA2/CTB chimera construct for maximal stability and antigenicity. The corresponding CfaE-A2/heat- labile enterotoxin (LT) B subunit chimera will then be made and compared to CfaE-A2/CTB in head-to-head immunogenicity studies in mice for purposes of downselection to the more immunogenic prototype chimera. This lead chimera vaccine component will be evaluated in non-human primates to determine a suitable dose and then to determine its protective efficacy using a monkey oral ETEC challenge model. Guided by these results, two additional sub-aims will be pursued. Using the optimized adhesin-toxoid scaffold, we will develop chimeras with two other ETEC fimbrial adhesins that together with CfaE would constitute a multivalent vaccine to confer broad-based protection. We will also scale up production processes and bring the lead CfaE-toxoid chimera through one campaign of cGMP manufacturing to lay the groundwork for preclinical and Phase I clinical trials in human subjects. Relevance. ETEC causes dehydrating diarrhea and poses a triple threat to potential victims of food bioterrorism, to military and civilian travelers, and to young children in resource-limited countries. This proposal aims to develop a vaccine that combines intestinal adhesins and a nontoxic component of the heat-labile enterotoxin (LT). Related forms of these two antigens have been shown to confer protection against disease in experimental settings. We posit that their combination into a single delivery module for oral administration will constitute a safe, effective vaccine for needle free, oral delivery.